Bulletin of Insectology 64 (Supplement): S75-S76, 2011 ISSN 1721-8861 Generation of a specific monoclonal recombinant antibody against ‘Candidatus Phytoplasma aurantifolia’ using phage display technology Fatemeh SHAHRIYARI 1,2 , Mohammad Reza SAFARNEJAD 1 , Masoud SHAMSBAKHSH 2 1 Agricultural Biotechnology Research Institute of Iran (ABRII), Karaj, Iran 2 Tarbiat Modarres University, Tehran, Iran Abstract Witches’ broom disease of lime (WBDL) is a destructive disease caused by ‘Candidatus Phytoplasma aurantifolia’ and is a limit- ing factor for lime production in Southern Iran. Conventional strategies for disease management have shown little success and new approaches based on genetic engineering need to be considered. Lack of natural resistance against phytoplasma diseases has highlighted the importance of alternative approaches and recombinant antibody mediated resistance is among them. The immu- nodominant membrane protein (IMP) is a major protein present on the surface of phytoplasma cells and is important for both di- agnostics and interactions with plant hosts and insect vectors. Generation of specific recombinant antibodies with high binding ability against IMP proteins is beneficial for both diagnostic purposes and development of recombinant antibody-mediated resis- tance against disease. Phage display is a powerful technology for generation of specific recombinant antibodies such as single chain variable fragment antibodies (scFv). This study describes generation of a specific scFv fragment through panning of naïve phage display libraries. For this aim, the gene encoding the IMP protein was isolated and cloned into a bacterial expression vector and recombinant IMP protein was expressed in bacterial cells and purified through affinity chromatography. Purified recombinant IMP protein was used for panning of naïve Tomlinson I and J scFv phage display libraries. Following three rounds of panning for selection and amplifi- cation of specific binders, capability of individual clones for production of specific scFvs was evaluated by an ELISA assay. The preliminary results showed generation of specific scFv recombinant antibodies with strong binding ability against the IMP pro- tein. Complementary studies revealed that the scFvs are able to bind native IMP and could detect the presence of phytoplasma in infected plants. Further immunoassay analysis confirmed that generated scFvs are able to detect epitopes along the IMP amino acid residues. As far as we know, this is the first succesful application of phage display libraries for generation of scFv recombi- nant antibodies against phytoplasma cells. Key words: phage display, WBDL, IMP, ‘Candidatus phytoplasma aurantifolia’, recombinant antibody, scFv. Introduction The witches’ broom disease of lime (WBDL) caused by ‘Candidatus Phytoplasma aurantifolia’, is the most de- structive disease in lime tree throughout southern Iran. Phytoplasmas are phloem-limited bacterial pathogens that persistently colonize their plant hosts. They are wall-less prokaryotes belong to Mollicutes. They are known to have specific characteristics such as small ge- nome size from 530 to 1350 Kb, low G+C percent, un- culturability in cell free media, and transmission and spread by insect vectors mainly leafhoppers and planthoppers (Lee et al., 2000). Phytoplasmas are surrounded by a single cell mem- brane. The Immunodominant membrane proteins (IMPs) of phytoplasmas are major proteins located on the external surface of the cell membrane and appear to have important roles in pathogenicity in the host plant and insect cells (Hogenhout et al., 2008). Due to the unique behaviour of IMP in pathogenecity of phyto- plasma, they are good candidates for suppression of dis- ease through a recombinant antibody-mediated resis- tance approach. Le Gall et al. (1998) generated an scFv fragment binding to the IMP protein of stolbur phyto- plasma. The plants expressing this scFv showed the po- tential for recombinant antibody fragments for suppres- sion of phytoplasma diseases in transgenic plants (Le Gall et al., 1998; Malembic-Maher et al., 2005). Phage display is a powerful technology for develop- ment of specific binders against almost any antigen. The main advantage of phage display is direct and physical linkage between phenotype and genotype. Several scFv phage display libraries have been developed by amplify- ing V L and V H regions of animal donors and fusing them to the pIII minor coat protein of a filamentous bacterio- phage (Hust and Dubel, 2004). This article introduces application of phage display technology for generation of a specific scFv recombinant antibody that could be used for both specific detection of WBDL infected plants and immunomodulation of disease in transgenic plants. Materials and methods The gene encoding the IMP protein was isolated and cloned into a bacterial expression vector and the puri- fied recombinant protein was obtained through affinity chromatography as previously described (Shahriyari et al., 2010). Phage display processes were carried out by performing three rounds of panning on Tomlinson I and J scFv phage display libraries as previously described (Safarnejad et al., 2008). Phytoplasma detection and characterization II